The core regulatory circuitry that drives and paces cell cycle progression in the bacterium Caulobacter crescentus constitutes a highly integrated system designed to ensure that multiple events take place in a spatiotemporally regulated manner. Although identification and characterization of various master regulators has offered extraordinary insight into the molecular basis of cell cycle control, the recent identification of novel, essential transcription factors and non-coding genetic elements in the Caulobacter genome strongly suggest an incomplete regulatory circuit and novel, undescribed regulatory modalities. The central goal of this project is to determine the roles of novel essential regulator elements in cell cycle control. Specifically, I propose to identify the regulatory functions of two novel cell-cycle-regulated Caulobacter transcription factors by using microarray analysis to identify genes regulated by each. I will employ ChIP-chip and bioinformatic analysis to experimentally and computationally identify the binding motif(s) for each transcription factor. In addition, I will conduct genetic and biochemical analysis to determine the requirement for essential non-coding chromosomal elements in cell cycle progression. Among the intergenic, non-coding essential gap sequences recently identified in the Caulobacter genome, over 60% lie immediately adjacent to cell-cycle-regulated genes, suggesting a role for these sequences in cell cycle control. Therefore, I will divide this subset into five categories based on the points during the cell cycle at which adjacent ORFs are induced and characterize representative sequences from each set through deletion, complementation, inversion, and transposition analysis. This will allow me to probe the essential nature of these sequences by manipulating various parameters such as length, position, and orientation. As preliminary evidence suggests that proteins may bind some of these sequences, I will use DNA sampling in order to crosslink and purify proteins that bind each sequence specifically. This broad approach will enable me to determine how the new essential elements of the Caulobacter genome are integrated into the core genetic circuit that controls cell growth and development.